The invention concerns a method for the temperature control of biological materials at sub-zero (below freezing) temperatures, particularly, a method for rapid internal cooling and rewarming of vascular organs and tissues with an inert fluid. This invention also relates to a class of new cryoprotective agents (CPA) used for organ cryopreservation and cryonics.
Biological processes can be greatly slowed at low temperatures, and even completely stopped at temperatures far below freezing. This principle is now successfully used to store at low temperatures (cryopreserve) and later recover a variety of living biological materials including cell cultures, blood, sperm, embryos, and skin. Chemical cryoprotective agents (CPAs) are added to the biological material prior to cooling to reduce ice formation and minimize the mechanical injury to the biological material caused by ice crystals formed during cooling.
One objective of organ cryopreservation is the long-term banking of transplantable organs at low temperatures until they are needed by recipients who are optimally matched for them. Technology to achieve this objective is the subject of ongoing research. Freezing and vitrification are two different approaches currently being pursued. For freezing, the organ to be cryopreserved is perfused with a sufficient concentration of cryoprotective agent so that ice formation is limited to non-lethal amounts during subsequent cooling. For vitrification, the organ is perfused with even higher concentrations of CPA so that ice formation is avoided completely on cooling (see U.S. Pat. No. 4,559,298, U.S. Pat. No. 5,217,860). In both cases, organs must be typically cooled to temperatures below -100.degree. C. for long-term stability.
Cryopreservation of organs or large organisms presents special problems as vascular tissues and the complex assemblages of cells which makes up the functional elements of parenchymatous organs (i.e. renal tubular architecture, neuronal interconnections and long processes) are more sensitive to injury by ice formation than small tissue samples or cell suspensions. Cryopreservation of vascularized tissue and solid organs with complex interdependent assemblages of cells requires replacement of a large fraction of cell and tissue water (usually by perfusion) with a high concentration of CPA to reduce ice formation to low levels, or to avoid ice formation completely by vitrification. This demanding application requires that CPAs be highly penetrating, non-toxic, and strongly inhibit ice formation. If vitrification is sought specifically, the CPA must also vitrify at low concentration, or be a "good glass former".
CPAs which have been explored for intracellular use in organ cryopreservation include dimethyl sulfoxide, alcohols, polyols (including ethylene glycol, propylene glycol, glycerol, butanediols), amides (including formamide, acetamide), and alkylamides (including methyl formamide, dimethyl formamide, diethylformamide). As yet, none of these agents or combination thereof have proven fully satisfactory for cryopreservation of large mammalian organs.
One objective of cryonics is to cryopreserve the entire human body or brain of a terminally-ill patient with sufficient fidelity that future medical technology might permit resuscitation and treatment. This objective is highly speculative at present. The long-term goal of cryonics research is the development of completely reversible means for maintaining humans in a state of arrested metabolism ("suspended animation"). The technologies and procedures used in cryonics are similar to those being developed for individual organ cryopreservation, except that they are applied to the whole body or brain.
For three decades, the CPA of choice in cryonics research has been glycerol. All the other agents tested proved to be excessively toxic to the central nervous system, causing edema and/or worsened ultrastructural preservation. However, the disadvantages of glycerol are numerous. It is viscous and does not penetrate mammalian cells readily at temperatures below 10.degree. C., necessitating perfusion at relatively high temperatures, thereby exacerbating ischemic injury. It permeates poorly, causing massive dehydration and poor preservation of myelinated areas of the central nervous system. It is a poor glass former, making it unsuitable for vitrification in concentrations which can be introduced by perfusion at acceptable temperatures.
It is an on-going effort to search for CPAs with high penetrating ability, low toxicity, low viscosity, strong freezing point depression, and good glass forming characteristics. The present invention discloses a class of new glycol ether cryoprotective agents with excellent overall properties.
The rate and uniformity with which tissues are cooled after perfusion with CPA are crucial parameters for both organ cryopreservation and cryonics. Cooling rate affects the distribution and size of ice crystals formed during freezing. Intercellular ice crystals formed during rapid cooling are typically smaller and less injurious than those formed during slow cooling. CPA toxicity is also greatest at high temperatures. Rapid cooling minimizes CPA exposure time at high sub-zero temperatures, thereby reducing toxic effects.
Rapid cooling is essential for vitrification. With rapid cooling not only are the toxic effects of the CPA minimized, but the concentration of CPA needed to vitrify is also reduced. Vitrification with non-lethal CPA concentrations typically requires cooling rates of several degrees per minute. Cooling uniformity is also important because vitrified organs may fracture if exposed to temperature gradients near their glass transition temperature.
In the prior art, at the completion of CPA perfusion, cooling is typically performed by immersing organs or cryonics patients in cold fluids for external cooling. Achievable cooling rates range from 10.degree. C. per minute for 8 ml samples (G. M. Fahy et al, Physical problems with the vitrification of large biological systems, Cryobiology 27, 492-510 (1990)) to less than 0.1.degree. C. per minute for cryonics patients (Cryonics: Reaching for Tomorrow, B. Wowk, M. Darwin, Alcor Life Extension Foundation, 3rd Edition, 1991.). Referring to 8 ml samples, Fahy et al write that larger samples must "inevitably" be cooled slower than 10.degree. C. per minute, and that this conclusion is "ominous" given the need for even higher CPA concentrations to vitrify at slower cooling rates, and the already borderline toxicity of CPA concentrations needed while cooling at 10.degree. C. per minute. Efforts for achieving vitrification of large organs by cooling methods of the prior art thus appear to be at an impasse.
Rapid rewarming is also beneficial for recovery from cryopreservation, as frozen tissue tends to recrystallize (grow larger damaging ice crystals) if it is rewarmed slowly. CPA exposure time, and associated toxicity is also reduced if rewarming is rapid. The rewarming requirements for vitrification are especially stringent, with heating rates greater than 100.degree. C. per minute typically required to avoid devitrification (ice formation) during rewarming. Specialized RF heating systems have been developed for this purpose.
It is clear from the above discussion that relatively high concentration CPAs are required for both freezing and vitrification in organ cryopreservation and cryonics, but high concentration CPAs are damaging to biological materials because of the inherent toxicity of the CPAs. Several general principles for reducing high concentration CPA damage are known which, for example, include exposure to the highest CPA concentrations at reduced temperature and time; the use of special combinations of CPAs and carrier solvents which cancel each other's toxicities; the use of nonpenetrating CPAs that can substitute for a portion of the penetrating agent otherwise needed (see U.S. Pat. No. 5,217,860). Among them, the most important and most effective is to increase cooling/rewarming rate, as discussed earlier, which reduces the exposure time to high concentration CPAs as well as significantly suppresses the formation of ice crystals.
Therefore, an objective of the present invention is to provide a method for internally and rapidly cooling/rewarming a biological material below the freezing point of water by circulating a cooled or warmed inert fluid through its vascular system, so that good cryopreservation can be achieved for organs or large organisms.
Another objective of the present invention is to provide a method for maintaining an organ at low temperature in situ to cryopreserve the organ without removal from a living animal.
Still another objective of the present invention is to provide a class of new glycol ether CPAs with special properties suitable for cryopreservation purposes.